A. The Unmet Need—Unprocessed Clinical and Forensic Samples
From the first isolation of nucleic acids by Miescher and Altmann in the second half of the nineteenth century (Miescher, Friedrich (1871) “Ueber die chemische Zusammensetzung der Eiterzellen,” in F. Miescher. Die Histochemischen and physiologischen Arbeiten Vol. 2:3-23) to the most sophisticated molecular biological techniques available today, the process of DNA extraction has been streamlined substantially. Nevertheless, there is a pressing need in the clinical, biothreat detection, and forensics communities for sensitive, robust, and reliable integrated methods of DNA purification that are rapid, cost-effective, and neither labor- nor space-intensive. In particular, there is an unmet need for methods and devices that can rapidly purify nucleic acids from unprocessed clinical or forensic field samples without any manual handling or processing.
Ideally, novel methods for nucleic acid purification are needed to address the numerous and varied existing and emerging markets for delivering genomic information, particularly the delivery of genomic information in the field, and for point-of-care and near point-of-care applications. For example, in the field of human identification, there is an unmet need in the forensic community to be able to generate a DNA fingerprint rapidly, whether in the laboratory or in the field (e.g. at borders, ports of entry, the battlefield, and military checkpoints).
Similarly, in order to protect civilian and military populations, it is critical to improve the identification of environmental biothreats. More rapid, more sensitive, more specific, and more detailed identification will allow improved strategic and tactical responses by civilian and military authorities, and more effective remediation activities. The rapid application of nucleic acid analysis technologies including nucleic acid amplification, hybridization, and sequencing can provide critical information in this regard.
Furthermore, the ability to rapidly diagnose clinical infections (whether caused by biothreats or conventional pathogens) would have a profound impact on society. For example, drawing a blood sample from a septic patient and determining both the identity of the pathogen or pathogens as well as their antibiotic resistance profiles based on nucleic acid analyses within an hour or less would allow specific antimicrobial therapy to begin immediately (the analogous situation for viral diagnostics and drug resistance profiles is also critically important). The ability to rapidly generate nucleic acid analytic information from clinical samples would also have substantial impact on the diagnosis and treatment of a wide range of diseases ranging from cancers to immune system disorders; essentially every category of diseases would be impacted. The same approach could also be applied to pharmacogenomics, the use of genetic information to predict the suitability of a given pharmacologic intervention.
B. Prior Art Approaches to DNA Purification
The basic approach to extraction and purification of nuclear DNA from mammalian cells was developed over three decades ago (N. Blin, D. W. Stafford (1976). A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 3(9): 2303-8) and has two major steps: the lysis of the cell types of interest and the purification of DNA from other cellular components in solution (particularly proteins) and cellular and tissue debris. Cell lysis and (when appropriate) DNA solubilization can be accomplished by mechanical (reviewed in J. Brent (1998). Breaking Up Isn't Hard To Do: A cacophony of sonicators, cell bombs and grinders” The Scientist 12(22):23) and non-mechanical techniques. Simple mechanical approaches include the use of a blenders and homogenization by forcing cells through restrictive openings. Sonication is based on the exposure of cells to high-frequency sound waves, and bead approaches are based on exposing cells to violent mixing in the presence of various beads.
Chemical disruption of cells is an alternative to mechanical disruption. Detergents are important chemical lytic agents that act by disrupting lipid bilayers. Additional properties of detergents may allow protein structure to be maintained (e.g. zwitterionic and nonionic detergents) or disrupted (ionic detergents). Sodium dodecyl sulfate (SDS), an ionic detergent, is commonly used in forensic DNA extraction protocols due in part to its ability to solubilize macromolecules and denature proteins within the cell (J. L. Haines et al (2005) Current Protocols in Human Genetics Vol. 2, (2005 John Wiley and Sons, Inc. Pub.). Proteinase K is often used in tandem with detergent-based (e.g. SDS, Tween-20, Triton X-100) lysis protocols. Another form of detergent lysis is based on FTA paper (L. A. Burgoyne (1997) Convenient DNA Collection and Processing: Disposable Toothbrushes and FTA Paper as a Non-threatening Buccal-Cell Collection Kit Compatible with Automatable DNA Processing, 8th International Symposium on Human Identification, Sept. 17-20, 1997 Orlando, Fla., G. M. Fomovskaia et al., U.S. Pat. No. 6,958,392). This is a cellulose filter impregnated with a weak base, an anionic detergent, a chelating agent, and preservatives.
In the case of a clinical or environmental sample, a critical first step towards nucleic acid analysis is the isolation or purification of some or all of the nucleic acid present in the sample. The biological material in the sample may be lysed and nucleic acids within the lysate may be purified prior to further analysis. Alternatively, nucleic acids contained within the lysate may be analyzed directly (e.g. Phusion Blood Direct PCR kit (Finnzymes, Espoo, FN) and Daniel et al., U.S. Pat. No. 7,547,510).
As those skilled in the art will recognize, purifying nucleic acids from unprocessed clinical, environmental, or forensic samples requires the automation of pre-processing steps suited to the particular field sample under investigation. The diversity of sample types, sample volumes, sampling technologies, sample collection devices, sample processing requirements, and the complexities inherent in resolving field samples has created an unmet need for robust methods and devices for purifying nucleic acids from such diverse samples.
C. Microfluidic Approaches to Purification from Clinical and Environmental Samples
The field of microfluidics offers a potential solution to the unmet need for methods and devices capable of isolating nucleic acids from unprocessed clinical, environmental, and forensic samples. Microfluidics is based on the manipulation of small fluid volumes of microliters or less and emerged as a hybrid of molecular biology and microelectronics in the early 1990's (See Manz et al. Sens. Actuators B1:244-248 (1990)). A major focus in microfluidics is to integrate multiple components to develop a system with sample-in, results-out functionality (reviewed in Erickson et al., Anal. Chimica Acta 507: 11-26 (2004)).
Some progress using this approach has been made with regard to environmental detection of biothreats. The automated pathogen detection system (Hindon et al., Anal. Chem. 77:284-289 (2005)) collects air samples and performs microfluidic DNA extraction and real-time PCR capable of detecting B. anthracis and Y. pestis (detection limits were between 103-107 organisms per mL of concentrated sample). The Cepheid (Sunnyvale, Calif.) GeneXpert system also collects air samples and performs integrated B. anthracis spore lysis (by microsonication), DNA extraction, and real-time PCR (detection limits were 68 cfu (equivalent to 148 spores) per mL concentrated sample for Ames spores and 102-103 cfu per mL concentrated sample for Sterne spores). Despite these advances, there is no available system or device capable of purifying unprocessed nucleic acids from clinical or environmental samples (or from environmental samples collected manually) without human intervention. Indeed, all of the available technologies rely on manual processing of some or all of the steps.
D. DNA Purification from Forensic Samples
One of the earliest DNA purification methods for forensic samples was the use of phenol/chloroform extraction (D. M. Wallace (1987) Large and small scale phenol extractions. Methods Enzymol. 152:33-41; Maniatis, T. et al., “Purification of Nucleic Acids” in Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). In this method, most protein moves to the organic phase or the organic-aqueous interface, and solubilized DNA remains in the aqueous phase. The DNA-containing phase can be subjected to ethanol precipitation, and DNA isolated following a series of centrifugation and wash steps. In forensic practice, DNA is often recovered from the aqueous phase with centrifugal dialysis devices, such as the Microcon columns (Millipore Corporation, Billerica, Mass.). The advantage of the organic extraction approach is that it yields high quality DNA preparations (with relatively low amounts of protein and relatively low degradation) and remains one of the most reliable methods available today. The major disadvantages are that the procedure is time- and labor-intensive, requires cumbersome equipment, and is relatively difficult to adapt to high-throughput settings.
Accordingly, the forensic community has moved to a series of purification technologies that are simpler to use, many of which serve as the basis of commercially available kits. There are an enormous number of approaches to nucleic acid purification, several of which are summarized as follows:
Silica Matrices/Chaotropic Agents.
The use of silica beads for DNA isolation has been a standard technique for over a quarter century, with the initial protocols based on the binding of DNA to silica in the presence of chaotropic agents such as sodium iodide (B. Vogelstein et al., (1979) “Preparative and analytical purification of DNA from agarose,” Proc Nat Acad Sci USA 76(2):615-9). Many years earlier, guanidinium salts had been found to be potent destabilizers of macromolecules (von Hippel P. H. et al., (1964) “Neutral Salts: The Generality of Their Effects on the Stability of Macromolecular Conformations.” Science 145:577-580). Certain guanidinium salts also have the advantage of deactivating nucleases (Chirgwin J. M. et al., (1979) “Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease,” Biochemistry 18(24):5294-9). These observations were synthesized by Boom (Boom, R. et al., (1990) “Rapid and Simple method for purification of nucleic acids,” J Clin Microbiol. 28(3):495-503), who, in effect, used two related properties of guanidinium salts. The first, the ability of the salts to lyse cells, and the second, the ability of the salts to enhance DNA binding to silica particles, have led to a number of lysis/purification approaches widely utilized in forensics laboratories today (e.g. DNAIQ Systems, Promega, Madison, Wis.). An alternative to silica beads is the use of silica membranes (QIAamp, Qiagen Hilden, Del.). In addition, the silica beads themselves may be modified to further enhance DNA binding.
Silica Matrices/Non-Chaotropic Agents.
Silica matrices can also be utilized in the absence of chaotropes. One approach is to modify silica beads such that they have a net positive charge at a given pH and are capable of binding DNA (Baker, M. J., U.S. Pat. No. 6,914,137). The modification contains an ionizable group, such that the DNA binding is reversed at a higher pH (when the ionizable group is neutral or negatively charged), sometimes at elevated temperature. As wide swings in pH can damage DNA, a critical feature of this approach is to choose a modification that allows reversible binding of DNA within a relatively narrow pH range. A widely used approach of this type is based on the ChargeSwitch bead (Life Technologies, Inc. Carlsbad, Calif.).
Magnetic Beads.
Although DNA binding properties are determined primarily by the surface structure of a given bead, the use of magnetic beads has become increasingly important in DNA purification protocols. These particles are generally paramagnetic; they are not themselves magnetic but form dipoles when exposed to a magnetic field. The utility of these beads relates to their ease of handling and adaptation to automated systems. For example, beads can be readily removed from a suspension in the presence of a magnet, allowing them to be washed and transported efficiently. Two commonly used magnetic beads are the ChargeSwitch and DNAIQ beads described above.
Ion Exchange.
Ion exchange allows DNA molecules to reversibly bind to an immobile bead. The bead generally consists of a porous organic or inorganic polymer with charged sites that allow one ion to be replaced by another at a given ionic strength. In practice, a solution containing DNA and other macromolecules is exposed to the ion exchange resin. The negatively charged DNA (due to its phosphate backbone) binds relatively strongly to the resin at a given salt concentration or pH. Protein, carbohydrate, and other impurities bind relatively weakly (if at all) and are washed from the beads (e.g. in a column format or by centrifugation). Purified DNA can then be eluted in a high ionic strength buffer. A commercially available anion exchange resin used today is based on DEAE-modified silica beads (Genomic-tip, Qiagen).
Chelex.
Chelex-100 (Bio-Rad, Hercules, Calif.). is a modified resin that efficiently binds multivalent metal cations. As such cations are required for enzymes that degrade DNA and themselves inhibit PCR enzymes, this method is representative of those that essentially avoid a DNA purification step (Walsh P. S. et al., Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 10(4):506-13).
When using cotton swabs to collect material, there can be problems removing biological material from the cotton matrix; as the cotton swab dries after collection, the biological material can adhere to the swab. For example, due to the saccharic composition of the spermatocyte membrane, spermatocytes stick to solid supports, especially cotton (Lazzarino, M. F. et al, (2008) DNA Recovery from Semen Swabs with the DNA IQ System. Forensic Science Communications 10(1)). In order to release the maximum amount of material from the swabs, a variety of buffers have been tested and compared to the standard differential extraction buffer. Use of detergents such as 1-2% sodium dodecyl sulfate (SDS) has shown to increase sperm cell recovery (Norris, J. V. et al., (2007) “Expedited, chemically enhanced sperm cell recovery from cotton swabs for rape kit analysis.” J Forensic Sci 52(4): 800-5). Also, the addition of low amounts of cellulase has shown to release more epithelial and sperm cells from the cotton swab matrix than buffer elution alone (Voorhees, J. C. et al., (2006). “Enhanced elution of sperm from cotton swabs via enzymatic digestion for rape kit analysis.” J Forensic Sci 51(3): 574-9).
There can be many challenges to obtaining forensic short tandem repeat (STR) profiles from biological materials including low quantity or quality of DNA. Low copy number samples (containing less than 50-100 picograms of DNA) as well as low quality, degraded samples require highly efficient collection, extraction, and amplification procedures. These samples are seen in a variety of forensic evidence including touch evidence and aged samples. Amplification kits such as the Life Technologies Minifiler™ have smaller amplicon sizes which have shown to increase the ability to obtain STR profiles from these difficult samples.
PCR inhibitors are another challenge and must be eliminated before downstream applications can be performed. Common inhibitors are indigo dyes from denim, heme from blood, humic acid found in plants and soil, and collagen found in various tissues. The majority of these inhibitors are effectively eliminated using silica based DNA extraction methods or additional purification with charge or size exclusion columns. The presence of inhibitors can be detected by performing PCR with internal positive controls. If present, some inhibitors can be neutralized by various treatments including sodium hydroxide washes or further purification with Millipore Microcon YM® columns.
The need to reconcile the “real world” requirements of sample collection with the microfluidic requirements of a fully integrated microfluidic DNA processing biochip can be referred to as the “macro-to-micro interface” or the “world-to-chip interface” (Fredrickson, C. and Fan, Z. (2004) “Macro-to-micro interfaces for microfluidic devices,” Lab Chip 4(6): 526-33). Much of the reported research on addressing this interface is focused on resolving the mismatch between the macrofluidic and microfluidic volumetric requirements, but little or no research concerning the reconciling of specific forensic sampling requirements and formats with microfluidic devices has been reported.
The (non-forensic) volumetric mismatch has been commercially addressed by Agilent (Santa Clara, Calif.) in the Bioanalyzer 2100 by the use of a capillary to aspirate samples from a microtiter plate to a chip for enzyme assays (Lin 2003). Similarly, Gyros (Uppsala, SE) has developed a capillary dispenser for a LabCD system where samples are aspirated from a well plate into a dispensing nozzle and then directed upwards onto a rotating device (Jesson 2003). These devices, however, do not address the format incompatibility of collected forensic samples—particularly on the commonly used collection devices based on swabs.
E. Partially Automated DNA Purification
A variety of laboratory instruments have been developed for the partially automated purification of nucleic acids. For example, the Maxwell 16 instrument (Promega) is designed to purify nucleic acids from forensic samples. To purify DNA from a buccal swab, the operator performs a number of steps including cutting the cotton collection portion in half, placing it into a 1.5 mL centrifuge tube, preparing and adding lysis reagents, incubating the sample in a heat block, vortexing the tube, transferring the reagents and swab sample to a spin basket, and centrifuging the basket. Next, a plunger is placed into the Maxwell cartridge, the sample is pipetted into the cartridge, and the cartridge is placed into the instrument for nucleic acid purification.
The iPrep instrument (Life Technologies) is also used for the processing of forensic and clinical samples to purify nucleic acids. For example, the tip of a buccal swab is placed into a 1.5 mL centrifuge tube and subjected to a series of manual steps similar to those required for the Maxwell 16. After manual sample preparation, the crude lysate is transferred to a 1 mL elution tube for processing within the instrument. The Qiagen EZ1, BioRobot M48, and Qiacube systems (Qiagen) partially automate nucleic acid purification. Buccal swabs are collected, allowed to dry for two hours, and manually processed essentially as with the instruments described above. Innuprep (analytikJena, Itzehoe, Del.), LabTurbo (Taigen, Taipei, T W), Xiril 150 (Xiril AG, Hombrechtikon, CH), and Quickgene (FujiFilm Corp., Tokyo, JP) systems are partially automated instruments requiring substantial user manipulation and intervention. U.S. Patent App. Pub. No. 20080003564 (Chen et al) describes a macrofluidic sample processing tube that accepts a swab and transports reagents mechanically using macrofluidic features and flexible tubing. US Patent App. Pub. No. 20070092901 (Ligler, F. et al.) have described a system that accepts liquid biological samples for semi-automated nucleic acid purification.
Several groups including those of Landers (Wolfe, K. A. et al., (2002) Toward a microchip-based solid phase extraction method for isolation of nucleic acids. Electrophoresis 23 (5):727-33; Wen, J. et al., (2006) DNA extraction using a tetramethyl orthosilicate-grafted photopolymerized monolithic solid phase. Anal Chem. 78(5):1673-81; Easley, C. J. et al., (2006) A fully integrated microfluidic genetic analysis system with sample-in-answer-out capability. Proc Natl Acad Sci USA 103(51):19272-7); Hagan K. A. et al. (2008) Microchip-based solid-phase purification of RNA from biological samples, Anal Chem 80:8453-60), Locascio (Becker, H. et al., (2002) Polymer microfluidic devices Talanta 56(2):267-287; Martynova, L. et al., (1997) Fabrication of plastic microfluid channels by imprinting methods. Anal Chem. 69(23):4783-9), Mathies (Lagally, E. T. et al., (2001) Fully integrated PCR-capillary electrophoresis microsystem for DNA analysis. Lab Chip 1(2):102-7: Yeung, S. H., et al., (2006) Rapid and high-throughput forensic short tandem repeat typing using a 96-lane microfabricated capillary array electrophoresis microdevice. J Forensics Sci. 51(4):740-7), and others (Liu R. H. et al., (2004) “Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction, amplification, and DNA microarray detection Anal Chem 76(7):1824-31) have been working on microfluidics for DNA purification and analysis (reviewed in Liu, P. and Mathies, R. A., (2009), “Integrated microfluidic systems for high-performance genetic analysis.” Trends in Biotechnology 27(10):572-81). Easley has demonstrated DNA isolation from 750 nanoliters of whole blood and 1 microliter of nasal aspirate using a guanidinium lysis/silica bead purification protocol (Easley, C. J. et al., Proc Natl Acad Sci supra). The whole blood sample contained approximately 2.5 million bacteria (Bacillus anthracis) per mL 1500-2000 cfu in the 750 nL sample), a concentration too high to be relevant for clinical diagnostics. U.S. Patent App. US2008/0014576 A1 describes nucleic acid purification modules that accept samples for purification in solutions, beads, colloids, or multiple-phase solutions and may be integrated with downstream preparation devices such as thermal cyclers and separation instruments.